This invention relates to culturing microorganisms in a culture vessel having both liquid and solid nutrient media. More particularly, it relates to a culture vessel in which two or more different solid nutrient media are easily separable from a liquid nutrient medium, which solid media can be selectively inoculated with the liquid nutrient medium without opening the vessel.
Microorganisms can be cultured either in a liquid nutrient medium, which is often referred to as a "broth," or on the surface of a nutrient medium which has been solidified with a gelling agent such as agar-agar, which medium is often referred to as either an "agar" or an "agar slant." Each culturing medium offers its own advantages. When cultured in a liquid nutrient medium, a single microbe will theoretically propagate a population of sufficient density to cause visible turbidity in the liquid medium within approximately 24 to 72 hours. When cultured on an agar medium, a single microbe of an input microorganism which is physically separate and immobile can develop into a visible colony which is clonogenic, quantifiable, and relatively easy to subculture. Since each of these types of culturing offers its own advantages, in many biological tests both culturing methods are employed By way of example, such culturing is commonly used when examining blood specimens for the presence of a variety of microorganisms, both aerobic and anaerobic, including brucella, salmonella, and many types of bacteria and fungi.
Typically, to detect microorganisms in a fluid sample, the sample is used to inoculate a liquid nutrient medium. Subsequently, the liquid medium is, in turn, used to inoculate a solid medium in order to grow a colony of the microorganisms. In order to obviate the need for accomplishing a troublesome and potentially dangerous transfer of the liquid medium preculture to the solid culture medium, from one container to another, culture vessels have been developed in which both the liquid and the solid culture media are contained in the same vessel. Because these vessels contain both solid and liquid media, they have come to be known as "biphasic." Generally, the sample to be tested is introduced into a liquid broth, and the culture vessel is then manipulated so as to mix the sample with the broth and also to wash the mixture over the solid nutrient medium surface. The vessel is usually then positioned so that the two media are separated and the media are incubated to allow colony growth. After a suitable incubation period, the solid and liquid nutrient media may be examined for the presence and/or growth of the suspect microorganisms.
An optimum biphasic culture vessel should meet several requirements and should provide solutions for a number of problems encountered by the culture vessels of the prior art The culture vessel must provide a means for keeping the liquid medium separated from the solid medium during transportation, storage, and use of the vessel, while at the same time allowing contact to be made between the liquid and solid media by appropriately manipulating the culture vessel. If the liquid and solid media are not separated during storage and use, the solid medium can dissolve into the liquid medium. At the very least, this mixing of the solid and liquid media can make differentiation of the pathogens difficult, and in some cases, mixing of the two media can produce hazardous conditions. At the same time, the culture vessel should be configured so as to facilitate microscopic examination of colonies growing on the solid nutrient medium, by looking through one or more of the culture vessel's walls. Also, the solid nutrient medium, being formed from a gelatinous material, is relatively easily dislodged from its mounting surface. Accordingly, the culture vessel should include means for retaining the solid nutrient medium in its proper position. Furthermore, the vessel should be designed so that the entire assembly can be sterilized by autoclaving after the solid and liquid nutrient media have been added to the vessel. For efficiency in performing multiple tests in a single vessel, it is also desirable that the culture vessel contain multiple solid nutrient media.
A number of culture vessels have been described in the prior art which address some of the requirements outlined above, but none of the vessels disclosed simultaneously provide all of these features U.S. Pat. No. 2,992,974, issued July 18, 1961 to A. S. Belcove et al., describes a biphasic culture vessel which includes a number of mechanisms for holding the agar slant in position within the vessel U.S. Pat. No. 3,532,605, issued Oct. 6, 1970 to J. V. Riera, describes a similar vessel in which a mesh is embedded in the solid nutrient material in order to further strengthen the agar slant. However, neither of the culture vessel assemblies described in those patents can be sterilized by autoclaving. For both of them, heating of the solid nutrient material would cause it to melt and become mixed with the liquid nutrient medium. Furthermore, for both of these culture vessel designs, the vessel must be placed in a generally horizontal position in order to separate the solid and liquid nutrient media during incubation. Then, in order to examine the surface of the solid nutrient medium for colony growth, the vessel must be placed in a generally upright position, thereby allowing the liquid medium to again contact the solid medium and to disturb the newly grown microorganism colonies.
U.S. Pat. No. 3,702,806, issued Nov. 14, 1972 to W. E. Oliva, discloses a culture vessel having a similar configuration to that described in the patent to Riera. In place of the cylindrical or rectangular vessel shown in the patent to Riera, the patent to Oliva discloses a vessel having a triangular cross-section. Oliva also discloses employing multiple agar slants, with different agars being mounted on each of the three surfaces which form the triangular cross-section. However, this culture vessel suffers the same inadequacies in sterilization capability and viewability as where noted above for the culture vessels described by Riera and Belcove et al.
A culture vessel which provides improved agar retention, media separability, and the capability of being sterilized by autoclaving is disclosed in U.S. Pat. No. 4,121,976, issued Oct. 24, 1978 to C. M. Gleeson. However, in the culture vessel shown in that patent, only one solid nutrient medium can be used in each vessel. The culture vessels described in U.S. Pat. No. 3,589,983, issued June 29, 1971 to W. J. Holderith et al., and in U.S. Pat. No. 4,308,347, issued Dec. 29, 1981 to H. Forrer et al., each may employ more than one solid nutrient medium in a single vessel. However, each of the vessels disclosed uses a two-piece design that is relatively complex and expensive to manufacture. The culture vessel of Forrer et al. also requires connecting the two portions of the vessel during use, which can easily lead to contamination of the culturing media.
What is needed, then, is a biphasic culture vessel which can employ two or more solid nutrient media, each of which can be inoculated separately by simple manipulation of the vessel. The liquid should be separated from the solid media during transportation, storage, and incubation. The vessel should be configured so that the entire assembly may be sterilized by an autoclaving process after the solid and liquid nutrient media have been deposited inside the vessel. The vessel should be further configured so that colonies of microorganisms growing on the solid nutrient media can be examined with an ordinary microscope. The vessel design should provide retention of the solid nutrient media in their positions within the vessel, while also facilitating subculturing of the microorganism colonies growing on the solid nutrient media. Finally, the vessel should be constructed of a relatively simple one-piece design which is inexpensive to manufacture and easy to use.
Accordingly, it is an object of the present invention to provide a biphasic culturing apparatus containing multiple solid nutrient media which are easily separable from the liquid nutrient medium during shipping, storage, and usage of the culturing apparatus.
It is also an object of the present invention to provide a biphasic culturing apparatus in which each solid nutrient medium can be separately inoculated by simple manipulation of the culturing apparatus, without opening the culture vessel.
It is another object of this invention to provide a culturing apparatus in which the solid nutrient media are sufficiently retained in position so that they are not dislodged during shipping or usage of the apparatus.
It is a further object of the instant invention to provide a culturing apparatus which is configured so that colonies of microorganisms growing on the solid nutrient media may be examined using an ordinary laboratory microscope, without opening the culture vessel.
It is still another object of the present invention to provide a biphasic culture vessel design which is simple and inexpensive to manufacture.
It is likewise an object of this invention to provide a method of detecting microorganisms in which multiple tests are concurrently conducted using a single culture vessel which contains multiple solid nutrient media.
It is another object of the present invention to provide a method for biphasic culturing of microorganisms wherein each solid nutrient medium may be separately inoculated with the liquid nutrient medium by tilting the culture vessel at a predetermined angle.
It is a further object of the instant invention to provide a method for preparing a biphasic culturing apparatus wherein the solid and liquid nutrient media may be sterilized either by aseptically depositing the media into a previously sterilized culture vessel, or by autoclaving the entire culturing apparatus after the solid and liquid nutrient media have been placed in the vessel.
It is also an object of the present invention to provide a method of culturing microorganisms in either an aerobic or an anaerobic environment.